The energetics of ligand-linked subunit assembly in hemoglobin require a third allosteric structure.
For partially ligated cyanomet hemoglobins, Smith and Ackers (Proc. Natl. Acad. Sci. U.S.A. 71 (1985) 4312) determined the free energies of dimer-tetramer assembly for all of the partially ligated species using a combination of kinetic and equilibrium methods. They found a third apparent cooperative free energy level in addition to those of deoxy- and cyanomethemoglobin. Using cryogenic methods, Perrella et al. (Biophys. Chem. 35 (1990) 97) confirmed the existence of the third cooperative free energy level, but found a different energy level assignment for one of the species. These combined studies have yielded a solid data base for considering mechanistic issues. The number of cooperative free energies delta Gc can, in principle, be different from the number of molecular forms which have unique free energies of heme-heme interaction, since delta Gc can be an average over conformational subspecies. Furthermore, since the delta Gc values are determined from free energies of dimer-tetramer assembly, it is necessary to evaluate possible contributions from dimeric properties, and from quaternary constraint (or enhancement) effects associated with subunit assembly. In this paper we analyze the observed distributions of apparent delta Gc values among the various ligation states in terms of mechanisms based on two interconvertible molecular forms (R and T) under the most general conditions in which (i) dimers may be cooperative, (ii) ligand affinities of alpha-subunits may be different within tetramers and dimers, and the same for beta-subunit affinities, and (iii) dimers need not be halves of R-state tetramers. It is found that the experimental distributions are inconsistent with even the most general model of the two-state class; thus, at least three molecular forms of tetramer are required, each with an individually different value of cooperative free energy (heme-heme interaction). This result implies the existence of at least three corresponding molecular structures; while a degeneracy of multiple structures into only a few dominant free energy levels is frequently to be expected, the reverse situation is extremely unlikely.
Ackers
G K
GK
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
D000494:Allosteric Regulation; D000495:Allosteric Site; D002151:Calorimetry; D006454:Hemoglobins / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008706:Methemoglobin / Q000031:analogs & derivatives / Q000378:metabolism; D008962:Models, Theoretical; D013816:Thermodynamics
D006454:Hemoglobins; D008024:Ligands; D046911:Macromolecular Substances; C007169:cyanomethemoglobin; D008706:Methemoglobin